The dark-coloured or melanic moths (carbonaria variety) were not known prior to 1811. After field collection in 1848 from Manchester, an industrial city in England, the frequency of the variety was found to have increased drastically. By the end of the 19th century it almost completely outnumbered the original light-coloured type (typica variety), with a record of 98% in 1895.[4] The evolutionary importance of the moth was only speculated upon during Darwin's lifetime. It was 14 years after Darwin’s death, in 1896, that J.W. Tutt presented it as a case of natural selection.[5]

Bernard Kettlewell was the first to investigate the evolutionary mechanism behind peppered moth adaptation, between 1953 and 1956. He found that a light-coloured body was an effective camouflage in a clean environment, such as in Dorset, while the dark colour was beneficial in a polluted environment like in Birmingham. This selective survival was due to birds which easily caught dark moths on clean trees, and white moths on trees darkened with soot. The story, supported by Kettlewell's experiment, became an example of Darwinian evolution used in standard textbooks.[6]

However, failure to replicate the experiment and criticism of Kettlewell's methods by Theodore David Sargent in the late 1960s led to general skepticism. When Judith Hooper's Of Moths and Men was published in 2002, Kettlewell's story was more sternly attacked, accused of fraud, and became widely disregarded. The criticism became a major argument for creationists. Michael Majerus was the principal defender. His seven-year experiment beginning in 2001, the most elaborate of its kind in population biology, the results of which were published posthumously in 2012, vindicated Kettlewell's work in great detail. This restored peppered moth evolution as "the most direct evidence", and "one of the clearest and most easily understood examples of Darwinian evolution in action".[7]

Typica and carbonaria morphs on the same tree. The light-coloured typica (below the bark's scar) is nearly invisible on this pollution-free tree, camouflaging it from predators.

Before the Industrial Revolution, the black peppered moth was rare. The first black specimen (of unknown origin) was kept in the University of Oxford in 1811.[8][9][10] The first live specimen was caught by R.S. Edleston in Manchester, England in 1848, but he reported this only 16 years later in 1864 in the journal Entomologist.[11] Edleston notes that by 1864 it was the more common type of moth in his garden in Manchester. The light-bodied moths were able to blend in with the light-coloured lichens and tree bark, and the less common black moth was more likely to be eaten by birds. As a result of the common light-coloured lichens and English trees, therefore, the light-coloured moths were much more effective at hiding from predators, and the frequency of the dark allele was about 0.01%.[12]

During the early decades of the Industrial Revolution in England, the countryside between London and Manchester became blanketed with soot from the new coal-burning factories. Many of the light-bodied lichens died from sulphur dioxide emissions, and the trees became darkened. This led to an increase in bird predation for light-coloured moths, as they no longer blended in as well in their pollutedecosystem: indeed, their bodies now dramatically contrasted with the colour of the bark. Dark-coloured moths, on the other hand, were camouflaged very well by the blackened trees.[6] The population of dark-coloured moth rapidly increased. By the mid-19th century, the number of dark-coloured moths had risen noticeably, and by 1895, the percentage of dark-coloured moths in Manchester was reported at 98%, a dramatic change (of almost 100%) from the original frequency.[6] This effect of industrialization in body colour led to the coining of the term "industrial melanism".[2]

The implications of industrial melanism to Charles Darwin's natural selection was evident during his lifetime. Albert Brydges Farn (1841–1921), a British entomologist, wrote to Darwin on 18 November 1878 to discuss his observation of colour variations in the Annulet moth (then Gnophos obscurata, now Charissa obscurata). He noted the existence of dark moths in peat in the New Forest, brown moths on clay and red soil in Herefordshire, and white moths on chalk cliffs in Lewes. He suggested this variation as an example of "survival of the fittest".[13] Surprisingly, scientific explanation came only in 1896, 14 years after Darwin’s death, when J.W. Tutt explicitly linked peppered moth melanism to natural selection. However, in a letter to Darwin, Farn explained his discovery of industrial melanism in Gnophos obscurata.[14] This letter shows that Darwin could have confirmed his theory of natural selection, had he realized the potential significance of the information that Farn provided.[15]

Melanism has appeared in the European and North American peppered moth populations. Information about the rise in frequency is scarce. Much more is known about the subsequent fall in phenotype frequency, as it has been measured by lepidopterists using moth traps.

Steward compiled data for the first recordings of the peppered moth by locality, and deduced that the carbonaria morph was the result of a single mutation that subsequently spread. By 1895, it had reached a reported frequency of 98% in Manchester.[16]

From around 1962 to the present, the phenotype frequency of carbonaria has steadily fallen. Its decline has been measured more accurately than its rise, because of more rigorous scientific studies being conducted. Notably, Bernard Kettlewell conducted a national survey in 1956, Bruce Grant conducted a similar one in early 1996,[17] and L.M. Cook in 2003.[18]

Similar results were found in America. Melanic forms have not been found in Japan. It is believed that this is because peppered moths in Japan do not inhabit industrialised regions.

In modern times, because of cleaner air standards in Europe and North America, the dark-bodied moth is becoming less frequent, again demonstrating the adaptive shifts in the peppered moth population.[19]

J.W. Tutt was the first to propose the "differential bird predation hypothesis" in 1896, as a mechanism of natural selection. The melanic morphs were better camouflaged against the bark of trees without foliose lichen, whereas the typica morphs were better camouflaged against trees with lichens. As a result, birds would find and eat those morphs that were not camouflaged with increased frequency.[20]

In 1924, J.B.S. Haldane calculated, using a simple general selection model, the selective advantage necessary for the recorded natural evolution of peppered moths, based on the assumption that in 1848 the frequency of dark-coloured moths was 2%, and by 1895 it was 95%. The dark-coloured, or melanic, form would have had to be 50% more fit than the typical, light-coloured form. Even taking into consideration possible errors in the model, this reasonably excluded the stochastic process of genetic drift, because the changes were too fast.[21] Haldane's statistical analysis of selection for the melanic variant in peppered moths became a well known part of his effort to demonstrate that mathematical models that combined natural selection with Mendelian genetics could explain evolution — an effort that played a key role in the foundation of the discipline of population genetics, and the beginnings of the modern evolutionary synthesis.[22]

In peppered moths, the allele for dark-bodied moths is dominant, while the allele for light-bodied moths is recessive, meaning that the typica moths have a phenotype (visible or detectable characteristic) that is only seen in a homozygousgenotype (an organism that has two copies of the same allele), and never in a heterozygous one. This helps explain how dramatically quickly the population changed when being selected for dark colouration.

The peppered moth Biston betularia is also a model of parallel evolution in the incidence of melanism in the British form (f. carbonaria) and the American form (f. swettaria) as they are indistinguishable in appearance. Genetic analysis indicates that both phenotypes are inherited as autosomal dominants. Cross hybridizations indicate the phenotypes are produced by isoalleles at a single locus.[23]

Several alternative hypotheses to natural selection as the driving force of evolution were proposed during the 1920s and 1930s. Random mutation, migration or genetic drift were also seen as major forces of evolution.[24] However, the magnitude of the changes observed can only be accounted for by natural selection. It can be seen from population genetics that a non-differential change will not cause evolution. If the allele frequencies are denoted by the algebraic terms p and q, and (say) p = 0.6 and q = 0.4, then a non-differential reduction in population size from say 2000 to 100 individuals, will still produce the same values of (approximately) p = 0.6 and q = 0.4.

P.A. Riley proposed an additional selective factor, where heavy metal chelation by melanin may protect peppered moths against the toxic effects of heavy metals associated with industrialisation. This selective advantage would supplement the major selective mechanism of differential bird predation.[25]

John William Heslop-Harrison (1920) rejected Tutt's differential bird predation hypothesis, on the basis that he did not believe that birds ate moths. Instead he advocated the idea that pollutants could cause changes to the soma and germ plasm of the organism. The origin of this hypothesis probably has its roots in the 1890s, when it was proposed as a form of Lamarckism. It is important to note its historical context.

Hasebroek (1925) was the first to try to prove this hypothesis. He contended that air pollution altered lepidopteran physiology, thus producing an excess of black pigment. He exposed pupae of Lepidoptera to various doses of pollutant gases, namely hydrogen sulfide (H2S), ammonia (NH3) and "pyredin" (presumably his spelling of pyridine). He used eight species in his studies, four of which were species of butterfly that did not exhibit melanism. Ford (1964) contends that Hasebroek's illustrations showed that the abnormal forms that appeared were not melanics, and Hasebroek failed to study their genetics.

Heslop Harrison (Harrison and Garrett 1926; Harrison 1928) suggested that the increase of melanic moths in industrialised regions was due to "mutation pressure", not to selection by predators which he regarded as negligible. Salts of lead and manganese were present in the airborne pollutant particles, and he suggested that these caused the mutation of genes for melanin production but of no others. He used Selenia bilunaria and Tephrosia bistortata as material. The larvae were fed with leaves that had incorporated these salts and melanics subsequently appeared.

Similar experiments by Hughes McKenney (1932) and Thomasen and Lemche (1933) failed to replicate these results. However, the statistician and geneticist Ronald Fisher showed that Heslop Harrison's controls were inadequate.[26] This hypothesis, however, appeared to be falsified by breeding experiments. Research into the biochemistry of melanism continues.

The first important experiments on the peppered moth were carried out by Bernard Kettlewell at Oxford University, under the supervision of E. B. Ford, who helped him gain a grant from the Nuffield Foundation to perform the experiments. In 1953, Kettlewell started a preliminary experiment in which moths were released into a large (18 x 6 m) aviary, where they were fed on by great tits (Parus major). His main experiment, at Cadbury Nature Reserve in Birmingham, England, involved marking, releasing and recapturing marked moths. He found that in this polluted woodland typica moths were preferentially preyed upon. He thus showed that the melanic phenotype was important to the survival of peppered moths in such a habitat. Kettlewell repeated the experiment in 1955 in unpolluted woodlands in Dorset and again in the polluted woods in Birmingham. He was accompanied by Nico Tinbergen, and they made a film together.[2][20] In 1956 he repeated the experiments and found similar results; in Birmingham birds ate most of the white moths (75%), whereas in Dorset most of the dark moths (86%) were eaten.[1][27]

Theodore David Sargent, professor of zoology at the University of Massachusetts at Amherst, published a critique of Kettlewell's work. Based on his experiments between 1965 and 1969, he concluded that it was not possible to reproduce Kettlewell's results, and said that birds showed no preference on moth on either black or white tree trunks.[28][29] He suggested that Kettlewell had trained the birds to pick moths on tree trunks to obtain desired results.[3][30]

Michael Majerus's 1998 book Melanism: Evolution in Action is an adaptation of Kettlewell's The Evolution of Melanism, which discussed criticisms of Kettlewell's original experimental methods.[20] When the biologist Jerry Coyne reviewed this book in Nature, he stated that the most serious problem was that only two peppered moths had been found on tree trunks. He also wrote that the white moths had increased in numbers before the lichen had returned and that Kettlewell's findings of moths choosing matching backgrounds had not been replicated in later experiments. Coyne compared his reaction to "the dismay attending my discovery, at the age of 6, that it was my father and not Santa who brought the presents on Christmas Eve". He concluded that "for the time being we must discard Biston as a well-understood example of natural selection in action, although it is clearly a case of evolution. There are many studies more appropriate for use in the classroom" and that further studies of the animal's habits were needed.[31]

Contrary to this review, Majerus had stressed that the basic findings from that work were correct, and that differential bird predation of polluted environment "is the primary influence of the evolution of melanism in the peppered moth".[32][33] Coyne's statement that only two peppered moths had been found on tree trunks was incorrect, as the book gives the resting positions of 47 peppered moths Majerus had found in the wild between 1964 and 1996; twelve were on tree trunks (six exposed, six unexposed), twenty were at the trunk/branch joint, and fifteen resting on branches.[32] Majerus found that the review did not reflect the factual content of the book or his own views,[34] and cited an assessment by the entomologistDonald Frack that there was essentially no resemblance between the book and Coyne's review,[35] which appeared to be a summary of the Sargent et al. paper rather than Majerus's book.[36]

The review was subsequently picked up by journalist Robert Matthews, who wrote an article for The Sunday Telegraph, 14 March 1999, claiming that "the rise and fall of the peppered moth, is based on a series of scientific blunders. Experiments using the moth in the Fifties and long believed to prove the truth of natural selection are now thought to be worthless, having been designed to come up with the 'right' answer." Majerus regarded this view as surprising, and not one that would be shared by those involved in the field. He noted numerous scientific inaccuracies, misquotations and misrepresentations in the article, but thought this was common in press reports.[35] He stated that he had spoken to Matthews for over half an hour and had to explain many details as Matthews hadn't read the book, but "Even then, he got nearly everything wrong."[34]

A 2002 book Of Moths and Men, by journalist Judith Hooper,[37] said Kettlewell's experiments had appeared to be "the slam-dunk of natural selection", but argued that the cause of the dark forms appearing was still an "irreducible mystery".[38] Although not a creationist herself, Hooper argued that peppered moth failed to represent evolution. She claimed that Kettlewell's field notes could not be found and suggested that his experiment was fraudulent, on the basis of Sargent's criticisms alleging that the photographs of the moths were taken of dead moths placed on a log. She said E.B. Ford was a "Darwinian zealot",[39] and claimed that he exploited the scientifically naive Kettlewell to obtain the desired experimental results. She then alleged that scientists in general showed "credulous and biased" acceptance of evolution.[40] The book's reception led to claims that the peppered moth evolution story ought to be deleted from textbooks.[41][42]

Scientists have examined the allegations made by Hooper, and found them to be without merit.[18][43][44] Majerus described the book as "littered with errors, misrepresentations, misinterpretations and falsehoods".[35] David W. Rudge, after critical analyses of the works of Kettlewell, declared that "none of Hooper's arguments is found to withstand careful scrutiny,"[45] and that all "these charges are baseless and stem from a fundamental misunderstanding of the nature of science as a process."[46] He concluded "that Hooper does not provide one shred of evidence to support this serious allegation”.[45]

Creationists have disputed the occurrence or significance of the melanic carbonaria morph increasing in frequency.

When serious criticism and controversy arose, the story was picked up by creationists. Coyne's review was taken up by intelligent design creationists, and at a seminar presenting the wedge strategy on 13 March 1999, creationist and professor of law Phillip E. Johnson said that the moths "do not sit on tree trunks", "moths had to be glued to the trunks" for pictures and that the experiments were "fraudulent" and a "scam."[47] This led Frack to exchange with intelligent design proponent Jonathan Wells, who conceded that Majerus listed six moths on exposed tree trunks (out of 47), but argued that this was "an insignificant proportion".[48] Wells wrote an essay on the subject, a shortened version of which appeared in The Scientist of 24 May 1999, claiming that "In 25 years of fieldwork, C.A. Clarke and his colleagues found only one peppered moth on a tree trunk", and concluding that "The fact that peppered moths do not normally rest on tree trunks invalidates Kettlewell's experiments".[49]

In 2000 Wells wrote Icons of Evolution, in which he claims, "What the textbooks don't explain, however, is that biologists have known since the 1980s that the classical story has some serious flaws. The most serious is that peppered moths in the wild don't even rest on tree trunks. The textbook photographs, it turns out, have been staged."[50] The arguments were dismissed by Majerus, Cook and Bruce Grant who describes Wells as distorting the picture by selectively omitting or scrambling references in a way that is dishonest.[32] Professional photography to illustrate textbooks uses dead insects because of the considerable difficulty in getting good images of both forms of moth in the same shot. The scientific studies actually consisted of observational data rather than using such photographs. The photographs in Majerus's Melanism: Evolution in Action are unstaged pictures of live moths in the wild, and the photographs of moths on tree-trunks, apart from some slight blurring, look little different from the "staged" photographs.[32] While an experiment did involve the gluing of dead moths to trees, this practice was just one of many different ways used to study different individual elements of the overall hypothesis. This particular experiment was not meant to exactly reproduce natural conditions but instead was used to assess how the numbers of moths available (their density) affected the foraging practices of birds.[51]

On 27 November 2000, the school board of Pratt County, Kansas continued efforts to favor intelligent design teaching by requiring the use of alternative resources, such as Of Pandas and People designed by Wells and other ID scholars.[52] (In the book Wells accused Kettlewell's experiment as "fraudulent" and "staged".)[53] Coyne and Grant wrote a letter to The Pratt Tribune in which they defended the moth experiments and revealed the misrepresentations by Wells.[54]

In 2000 Majerus developed plans for experiments to resolve the various valid criticisms of Kettlewell’s experiment. During his experiment in Cambridge over the six years 2001–2007 he noted the natural resting positions of peppered moths. Of the 135 moths examined over half were on tree branches, mostly on the lower half of the branch, 37% were on tree trunks, mostly on the north side, and only 12.6% were resting on or under twigs. Following correspondence with Hooper he added an experiment to find if bats, not birds, could be the main predators. He observed a number of species of bird actually preying on the moths, and that differential bird predation was a major factor responsible for the decline in carbonaria frequency compared to typica.[20] He described his results as a complete vindication of the natural selection theory of peppered moth evolution, and said "If the rise and fall of the peppered moth is one of the most visually impacting and easily understood examples of Darwinian evolution in action, it should be taught. It provides after all the proof of evolution."[55]

Majerus died before he could complete the writing up of his experiments, so the work was carried on by Cook, Grant, Saccheri and Mallet, and published on 8 February 2012 as "Selective bird predation on the peppered moth: the last experiment of Michael Majerus."[56] The experiment became the largest ever in the study of industrial melanism, involving 4,864 individuals in a six-year investigation, and it confirmed that melanism in moths is a genuine example of natural selection involving camouflage and predation. Their concluding remark runs: "These data provide the most direct evidence yet to implicate camouflage and bird predation as the overriding explanation for the rise and fall of melanism in moths."[7]

Coyne responded by saying, "Despite the defensiveness of British evolutionists, I think my criticisms carried some weight, because Cambridge biologist Michael Majerus decided to repeat Kettlewell's experiments, but doing them correctly this time." He quoted the Cook et al. conclusion that "These new data answer criticisms of earlier work and validate the methodology employed in many previous predation experiments that used tree trunks as resting sites. The new data, coupled with the weight of previously existing data convincingly show that 'industrial melanism in the peppered moth is still one of the clearest and most easily understood examples of Darwinian evolution in action'." Coyne said he was "delighted to agree with this conclusion, which answers my previous criticisms about the Biston story."[57]

On 19 June 2009, Telegraph.co.uk published an article on this evolutionary phenomenon and implored UK readers to visit the Moths Count website and record their observations of local moths, in an effort to help increase the available data for researchers.